Marine Diatom Cylindrotheca Research Articles

Investigation of the role of cell hydrophobicity and EPS production in the aggregation of the marine diatom Cylindrotheca closterium under hypo-saline conditions

Aggregation of diatoms is of global importance to understand settling of particulate organic carbon in aquatic systems. In this study, we investigate the aggregation of the marine diatom Cylindrotheca closterium during the exponential growth phase under hypo-saline conditions. The results of the flocculation/flotation experiments show that the aggregation of the diatom depends on the salinity. In favorable growth conditions for marine diatoms (salinity of 35), the highest aggregation is achieved. To explain these observations, we used a surface approach combining atomic force microscopy (AFM) and electrochemical methods to characterize both the cell surface properties and the structure of the extracellular polymeric substances (EPS) cell produce, and to quantify the amount of surface-active organic matter released. At a salinity of 35, the results showed that diatoms are soft, hydrophobic and release only small amounts of EPS organized into individual short fibrils. In contrast, diatoms adapt to a salinity of 5 by becoming much stiffer and more hydrophilic, producing larger amounts of EPS that structurally form an EPS network. Both adaptation responses of diatoms, the hydrophobic properties of diatoms and the release of EPS, appear to play an important role in diatom aggregation and explain the behavior observed at different salinities. This biophysical study provides important evidence allowing to get a deep insight into diatom interactions at the nanoscale, which may contribute to a better understanding of large-scale aggregation phenomena in aquatic systems.

Interaction of Silica Nanoparticles with Microalgal Extracellular Polymers

The properties of engineered nanoparticles (NPs) in the marine environment are influenced not only by the high ionic strength of seawater but also by the interaction of NPs with naturally occurring components of seawater, especially natural organic matter. The aim of this study was to investigate the interaction of engineered silica nanoparticles (SiO2 NPs, diameter of 12 nm) with microalgal extracellular polymers (EPS) released by the marine diatom Cylindrotheca closterium. Dissolved organic carbon (DOC) content of the prepared EPS suspension (200 μg mL−1) used throughout the study was 3.44 mg C L−1. The incorporation of individual SiO2 NPs (height range 10–15 nm) and their nanoscale aggregates (height up to 25 nm, length up to 600 nm) into the EPS network was visualized by atomic force microscopy (AFM), whereas their molecular-level interaction was unraveled by the change in the signal of the Si-O group in their FTIR spectra. AFM imaging of C. closterium cells taken directly from the culture spiked with SiO2 NPs (10 μg mL−1) revealed that the latter are bound to the EPS released around the cells, predominantly as single NPs (height range 10–15 nm). Since AFM and dynamic and electrophoretic light scattering results demonstrated that SiO2 NPs dispersed in seawater without EPS showed enhanced aggregation (aggregate diameter of 990 ± 170 nm) and a 2.7-fold lower absolute zeta potential value compared to that measured in ultrapure water, our findings suggest that the presence of EPS biopolymers alters the aggregation affinity of SiO2 NPs in the marine environment. This might be of outmost importance during microalgal blooms when increased EPS production is expected because EPS, by scavenging and stabilizing SiO2 NPs, could prolong the presence of NPs in the water column and pose a threat to marine biota.

Nanoplastic-Induced Nanostructural, Nanomechanical, and Antioxidant Response of Marine Diatom Cylindrotheca closterium

The aim of this study was to examine the effect of positively charged (amine-modified) and negatively charged (carboxyl-modified) polystyrene nanoplastics (PS NPs) on the nanostructural, nanomechanical, and antioxidant responses of the marine diatom Cylindrotheca closterium. The results showed that both types of PS NPs, regardless of surface charge, significantly inhibited the growth of C. closterium during short-term exposure (3 and 4 days). However, longer exposure (14 days) to both PS NPs types did not significantly inhibit growth, which might be related to the detoxifying effect of the microalgal extracellular polymers (EPS) and the higher cell abundance per PS NPs concentration. The exposure of C. closterium to both types of PS NPs at concentrations above the corresponding concentrations that resulted in a 50% reduction of growth (EC50) demonstrated phytotoxic effects, mainly due to the excessive production of reactive oxygen species, resulting in increased oxidative damage to lipids and changes to antioxidant enzyme activities. Diatoms exposed to nanoplastics also showed a significant decrease in cell wall rigidity, which could make the cells more vulnerable. Atomic force microscopy images showed that positively charged PS NPs were mainly adsorbed on the cell surface, while both types of PS NPs were incorporated into the EPS that serves to protect the cells. Since microalgal EPS are an important food source for phytoplankton grazers and higher trophic levels, the incorporation of NPs into the EPS and interactions with the cell walls themselves may pose a major threat to marine microalgae and higher trophic levels and, consequently, to the health and stability of the marine ecosystem.

First identification and characterization of detoxifying plastic-degrading DBP hydrolases in the marine diatom Cylindrotheca closterium

Diatoms are photosynthetic organisms with potential biotechnological applications in the bioremediation sector, having shown the capacity to reduce environmental concentrations of different pollutants. The diatom Cylindrotheca closterium is known to degrade di-n-butyl phthalate (DBP), one of the most abundant phthalate esters in aquatic environments and a known endocrine-disrupting chemical. In this study, we present for the first time the in silico identification of two putative DBP hydrolases (provisionally called DBPH1 and DBPH2) in the transcriptome of C. closterium. We modeled the structure of both DBPH1–2 and their proposed interactions with the substrate to gain insights into their mechanism of action. Finally, we analyzed the expression levels of the two putative hydrolases upon exposure of C. closterium to different concentrations of DBP (5 and 10 mg/l) for 24 and 48 h. The data showed a DBP concentration-dependent increase in expression levels of both dbph1 and 2 genes, further highlighting their potential involvement in phthalates degradation. This is the first identification of phthalate-degrading enzymes in microalgae, providing new insights into the possible use of diatoms in bioremediation strategies targeting phthalates.

In silico first identification and modelling of DBP hydrolases in the marine diatom Cylindrotheca closterium

In silico first identification and modelling of DBP hydrolases in the marine diatom Cylindrotheca closterium

The role of substrates towards marine diatom Cylindrotheca fusiformis adhesion and biofilm development

The role of substrates towards marine diatom Cylindrotheca fusiformis adhesion and biofilm development

Physiological and morphological response of marine diatom Cylindrotheca closterium (Bacillariophyceae) exposed to Cadmium

ABSTRACT The impact of the heavy metal cadmium on the lightly silicified marine diatom Cylindrotheca closterium was studied with regard to growth dynamics, organic matter production and morphological characteristics. C. closterium cultures were able to sustain positive growth in the tested range of 0–1000 μg Cd l–1 with a significant decrease in specific growth rate in the exponential growth phase at the highest tested Cd concentration. The concentrations of both dissolved (DOCcell) and particulate organic carbon per cell (POCcell) in C. closterium cultures exposed to 500 and 1000 µg l–1 Cd more than doubled compared with the control culture. Enhanced organic matter production as a feedback response to Cd exposure may thus contribute to cell detoxification, supported by the electrochemical data showing that Cd was not significantly accumulated by or associated with diatom cells except for the culture exposed to 1000 μg Cd l–1. In order to gain more insight into the effects of cadmium on diatom morphological features, the cell surface of C. closterium was characterized at the nanoscale on a single cell level using atomic force microscopy (AFM). Changes induced by Cd were observed as irregular patterns of silica spheres on more silicified parts of the cell, girdle band and around the raphe. These changes clearly show that beside the physiological response, C. closterium also showed a morphological response which is probably due to the interference of Cd with the diatom biosilification process. This study overall contributes to better understanding of the effects of cadmium on diatoms and shows that morphological characteristics assessed by AFM may be a valuable indicator for metal contamination.

Production of Surface Active Organic Material and Reduced Sulfur Species During the Growth of Marine Diatom Cylindrotheca closterium

Electrochemical methods at the mercury electrode were used for monitoring production of surface active substances (SAS) and reduced sulfur species (RSS) during growth of marine diatom Cylindrotheca closterium isolated from the Adriatic Sea in the laboratory conditions. In the same culture samples, production of particulate and dissolved organic carbon (POC, DOC) was followed by high temperature catalytic oxidation method (HTCO). The culture growth curve obtained by microscopically counted phytoplankton cells showed an exponential growth phase that lasted 10 days, transition phase until 14 days and stationary phase until 21 days. In these time periods twofold increase of SAS and DOC was followed, while POC increased 41 times. Detail analyses of a.c. out of phase voltammetric curves recorded in original and in acidified phytoplankton culture samples indicate transformation of organic material during growth, from more anionic (negatively charged) to less anionic polymeric surface active material. In culture samples presence of non-volatile RSS were confirmed.

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY(1).

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young's modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young's modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.

AFM imaging of extracellular polymer release by marine diatom Cylindrotheca closterium (Ehrenberg) Reiman & J.C. Lewin

Extracellular polysaccharide production by marine diatoms is a significant route by which photosynthetically produced organic carbon enters the trophic web and may influence the physical environment in the sea. This study highlights the capacity of atomic force microscopy (AFM) for investigating diatom extracellular polysaccharides with a subnanometer resolution. Here we address a ubiquitous marine diatom Cylindrotheca closterium, isolated from the northern Adriatic Sea, and its extracellular polymeric substance (EPS) at a single cell level. We applied a simple procedure for AFM imaging of diatom cells on mica under ambient conditions (in air) to achieve visualization of their EPS with molecular resolution. The EPS represents a web of polysaccharide fibrils with two types of cross-linking: fibrils association forming junction zones and fibril-globule interconnections with globules connecting two or more fibrils. The fibril heights were 0.4-2.6 nm while globules height was in the range of 3-12 nm. Polymer networks of native gel samples from the Northern Adriatic and the network formed by polysaccharides extracted from the C. closterium culture share the same features regarding the fibril heights, pore openings and the mode of fibril association, proving that the macroscopic gel phase in the Northern Adriatic can be formed directly by the self-assembly of diatom released polysaccharide fibrils.

Ring test for whole-sediment toxicity assay with -a- benthic marine diatom

This work presents the results of an interlaboratory proficiency exercise for whole-sediment toxicity assays with the benthic marine diatom Cylindrotheca closterium. An assay protocol was established and followed by all participating laboratories. Cell growth after 72 h exposure was the endpoint used. Four sediment samples of unknown toxicity were assayed. The main problem encountered during this exercise was the differences in the cell growth of algae exposed to reference sediment. Those differences may be associated with changes in the physiological status of the initial culture due to temperature changes during transport to the other laboratories. In general, the method proposed presented good replicability (precision between replicates) and reproducibility (interlaboratory precision). Around 80% (17 out of 21) of results obtained were classified as satisfactory ( Z-scores < 2). The whole-sediment assay with C. closterium presented here can be considered sufficiently successful for possible use as a standard toxicity test. The assay is simple to perform, the proposed species is ecologically relevant as an integral component of microphytobenthos, and is widely distributed around the world. These positive factors suggest that the whole-sediment assay with the benthic marine diatom C. closterium can be used as a reliable tool in marine sediment quality assessment.

UV- and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion

The diatom Cylindrotheca closterium was exposed to transient light- and osmotic conditions as occur during its tidal emersion. The objective was to analyze how this simulated emersion contributes to the production of active oxygen species (AOS) and via this, to oxidative cell damage. Light- and salinity conditions were varied in factorial combination: low light (no UVB) or high light (unweighted UVB-dose rates of respectively 0.01; 0.07; 0.24; 1.03 W m−2) at normal (30 psu) or high salinity (60 psu). UVB (0.01–0.24 W m−2) and high salinity had a significant, negative effect on the photosynthetic efficiencies ΔF/F m’ (steady-state quantum yield) and F v/F m (maximum yield). UVB at 1.03 W m−2 (15 kJ m−2 d−1) almost arrested electron transport. At ecologically relevant UVB levels, i.e. below 0.24 W m−2 (≈3.4 kJ m−2 d−1) with UVB:PAR 0.07 W m−2 and at 60 psu indicated a reversal of the diatom xanthophyll cycle (diminished photoprotection) which may be caused by an enhanced AOS production. Oxidative stress and -damage to C. closterium cells were assessed applying fluorescent indicator dyes, via confocal microscopy and quantitative image analysis. AOS production rates (cellular DCF fluorescence) were stimulated by UV, and were ~50% higher at 60 psu. AOS production decreased with an increasing pre-exposure (0–4 h) to normal UVB (0.24 W m−2), which indicated a stimulation of the antioxidative defence. Non-protein thiols (indicator CMF) and glutathione pools (HPLC-analyzed) decreased with UVB-dose rates (0.01–0.24 W m−2), most likely due to AOS-mediated thiol oxidation. Hypersalinity (60 psu) and UVB (0.01–0.24 W m−2) caused membrane depolarization (dye DIBAC4(3)) and phospholipid hydrolysis (phospholipase A2 dye: bis-BODIPY FL-C11-PC). AOS production may have diminished the membrane polarity, and peroxidized the membrane lipids (HPLC-analyzed malondialdehyde) which enhanced PLA2 activity. The dyes indicated an increased oxidative (lipid) damage at a 15% inhibition of photosynthesis in this diatom, at UVB levels and salinities that can be expected in situ during its periodic tidal emersion.

Regulation of the Si and C uptake and of the soluble free-silicon pool in a synchronised culture of Cylindrotheca fusiformis (Bacillariophyceae): effects on the Si/C ratio

Silicon and carbon uptake rates were studied over a 24 h light/dark cycle in a synchronised culture of the marine diatom Cylindrotheca fusiformis (Reimann et Lewin) using 32Si and 14C. The silicic acid uptake rate per cell (ρcSi) varied between 1.2 and 20.0 fmol Si cell−1 h−1 and was closely correlated to the G2+M phase of the cell cycle. A linear and significant relationship was determined between the percentage of cells present in G2+M and ρcSi. Evolution of the soluble free-silicon pool was studied simultaneously. The concentration of the total soluble free pool of silicon (QPSi) varied from 1% to 7% of the total silicon content. A significant difference of 1.5 fmol Si cell−1 between QPSi and the labelled free pool (QnpSi) was measured, indicating the presence of an unlabelled fraction of the pool. The concentration of QnpSi was around 1.0 fmol Si cell−1 prior to cell division and did not change as a function of ρcSi, which indicated a feedback mechanism coupling uptake into the free pool and incorporation into the frustule. In parallel, 14C uptake variation (ρcC) was measured during the division of the population. The value of ρcC varied between 0.44 and 0.78 pmol C cell−1 h−1 and appeared to be maximal when cells were in the G1 phase. This variation of ρcC marginally affected the total carbon content of the cells (QTC) in comparison with the light/dark cycle. The variations in the Si/C ratio, from 0.021 to 0.046, demonstrated the different control mechanisms of Si and C metabolisms during the course of the cell- and photocycle.

Abundance and chemical characterization of extracellular carbohydrates released by the marine diatom Cylindrotheca fusiformis under N- and P-limitation

The effect of P- and N-depletion on the production and molecular-level composition of extracellular carbohydrates released by the diatom Cylindrotheca fusiformis was examined in axenic batch cultures. Inorganic phosphorus depletion caused a higher release of organic carbon per cell compared to nutrient-replete and nitrogen-limited conditions. Gas-chromatographic analysis of the monosaccharide composition of the dissolved fraction showed galactose to be the major component of the exopolymers (33 – 42%). In the P-limited treatment, an increase of galactose content and a decrease of glucose was found, while the composition of the remaining saccharides was almost unaffected. The results point out the relevance of phosphorus limitation not only in causing an increase in total exopolysaccharide production, but also in affecting the neutral aldose composition of the dissolved carbohydrates released by C. fusiformis, with heteropolysaccharides being more abundant than glucan. The overall sugar composition of the exopolymers released under the most severe P-depletion was similar to that of natural mucilage samples collected in the Northern Adriatic Sea during the Summer 2000 event of massive aggregation.

Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium

During emersion, diatoms are exposed to sunlight (with UVB) on tidal flats, where the salinity may increase as a result of evaporation. In order to investigate whether a combination of UV radiation and desiccation would cause oxidative stress under such conditions, an experiment has been carried out with the diatom Cylindrotheca closterium. Cell division rates, photosynthetic efficiencies, pigment contents, and activities of antioxidant enzymes (SOD, APX, MDHAR, GR) were analysed in exponential-phase batch cultures grown at 35 (normal salinity) and 70 PSU (hyperosmotic) in a 12 h:12 h light:dark cycle (268 µmol photons m-2 s-1 photosynthetically active radiation; PAR) at 15°C. Within the 12 h photoperiod, UVA (2.80 W m-2 unweighted) or UVA+UVB (4.23 and 0.22 W m-2, respectively) were supplemented during 4 h. Separately as well as in combination, UVB (3.45 kJ m-2 d-1) and salt stress (70 PSU) caused a decrease in division rates. This UVB dose caused a decrease in photosynthetic efficiency, whereas salt stress did not. Cell volumes of UVB-exposed C. closterium increased only at 70 PSU. UVB radiation and salt stress caused significant decreases in chl a, chl c and fucoxanthin contents, but the UVB effect was stronger than the salt effect. The relatively high b-carotene:chl a ratios in UVB-exposed and salt-stressed cells might indicate that b-carotene was used in scavenging singlet oxygen. In high light with UVA (at 35 PSU), SOD, APX, MDHAR and GR activities were not higher than in low light (27 µmol photons m-2 s-1). Separately and in combination, UVB and salt stress enhanced SOD activity in C. closterium, whereas APX was stimulated by UVB only. MDHAR was stimulated under UVB and salt stress, but there were no effects on GR activity. Under ambient salt and UV conditions during emersion, oxidative stress may contribute to an inhibition of growth of C. closterium [KEYWORDS: Antioxidants; Cylindrotheca closterium; Diatom; Pigments; Salt stress; Ultraviolet]

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.

Silicon-responsive cDNA clones isolated from the marine diatom Cylindrotheca fusiformis

In organisms ranging from single-celled algae to mammals, including humans, silicon is essential for, and actively participates in, a variety of life processes. It has become clear that silicon (i) acts as a metabolite affecting a variety of cellular processes, and (ii) regulates gene expression. However, the mechanisms by which silicon (i.e., Na 2SiO 3-9H 2O, in the present study) acts are not clear, due to inherent methodological difficulties. As part of our program to understand how silicon acts in biological systems, we present the first isolation of cDNA clones derived from silicon-responsive mRNAs, from the marine diatom Cylindrotheca fusiformis. We distinguish between clones responding only to silicon starvation and replenishment, and those also responding to other cellular conditions. Some of the clones can be identified by similarity to other genes, and should be useful as probes to isolate genes from other organisms. Isolation of these clones provides the means to (i) identify metabolic pathways affected by silicon, and (ii) investigate the mechanism(s) of silicon-regulated gene expression.

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles.

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases.

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28-31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.

Characterization of two circular plasmids from the marine diatom Cylindrotheca fusiformis: plasmids hybridize to chloroplast and nuclear DNA.

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.